Covalent modification by isoprenoid lipids (prenylation) contributes to membrane interactions and biological activities of a rapidly expanding group of proteins (Maltese, FASEB J. 4:3319 (1990); Glomset et al, Trends Biochem. Sci., 15:139 (1990)). Either farnesyl (15-carbon) or geranylgeranyl (20-carbon) isoprenoids can be attached to specific proteins, with geranylgeranyl being the predominant isoprenoid found on proteins (Farnsworth et al, Science, 247:320 (1990)). The prenyltransferase, protein geranylgeranyltransferase type-I (GGTase-I), transfers a geranylgeranyl group from the prenyl donor geranylgeranyl diphosphate to the cysteine residue of substrate proteins containing a C-terminal CAAX-motif in which "A" is any amino acid, including an aliphatic amino acid, and the "X" residue is leucine (Clarke, Ann. Rev. Biochem., 61:355 (1992); Casey, J. Lipid. Res., 330:1731 (1992)). Known targets of GGTase-I include gamma-subunits of brain heterotrimeric G proteins and Ras-related small GTP-binding proteins such as Rac1, Rac2, Rap1A and Rap1B (Menard et al, Eur. J. Biochem., 206:537 (1992); Casey et al, Proc. Natl. Acad. Sci, USA, 88:8631 (1991); Moores et al, J. Biol. Chem., 136:14603 (1991)).
Additionally, short peptides encompassing the CAAX motif of these substrates can also be recognized by the enzyme (Casey et al, Proc. Natl. Acad. Sci. USA, 88:8631 (1991); (Moores et al, J. Biol. Chem., 136:14603 (1991); Yokoyama et al, Proc. NatL. Acad. Sci. USA, 88:5302 (1991)). Immobilization of one such peptide for use as an affinity matrix has led to the isolation of GGTase-I from bovine brain (Moomaw and Casey, J. Biol. Chem., 267:17438 (1992)). The purified enzyme contains two subunits with molecular masses of 48 kDa and 43 kDa, which have been designated, respectively, as alpha and beta (henceforth designated beta [GGI]). GGTase-I is dependent on both Mg.sup.2+ and Zn.sup.2+ for optimal activity. Demonstration of the Zn.sup.2+ dependence required prolonged incubation against, or purification in the presence of, a chelating agent. This property has led to the designation of GGTase-I as a zinc metalloenzyme (Moomaw and Casey, J. Biol. Chem. Id.).
The properties of GGTase-I are similar to those of a related enzyme, protein farnesyltransferase (FTase). FTase transfers the prenyl moiety from farnesyl diphosphate to the cysteine residue of substrate proteins. FTase protein substrates, like those for GGTase-I, possess a C-terminal CAAX motif. The "X" residue of mammalian FTase substrates, however, is generally methionine, serine or glutamine as opposed to leucine for GGTase-I substrates (Moores et al, J. Biol. Chem., 136:14603 (1991); Moomaw and Casey, J. Biol. Chem., 267:17438 (1992)). Substrates for FTase include p21 (ras) protein, lamin B and several proteins involved in visual signal transduction (Clarke, Ann. Rev. Biochem., 61:355 (1992)). Like GGTase-I, FTase is dependent upon Mg.sup.2+ and Zn.sup.2+ ions for optimal activity (Reiss et al, J. Biol. Chem., 267:6403 (1992)).
Purified mammalian FTase is composed of two nonidentical subunits, alpha and beta (henceforth designed beta F), with apparent molecular masses of approximately 48 kDa and 46 kDa, respectively, on SDS-PAGE (Reiss et al, Cell 62:81 (1990)). cDNA clones encoding the FTase alpha and beta F subunits have been isolated and their deduced amino acid sequences are homologous to the Saccharomyces cerevisiae proteins Ram2 and Dpr1/Ram1, respectively, which encode the subunits of yeast FTase (Moores et al, J. Biol. Chem., 136:14603 (1991); Chen et al, Proc. Natl. Acad Sci. USA, 88:11368 (1991); Kohl et al, J. Biol. Chem., 266:18884 (1991); He et al, Proc. Natl. Acad. Sci. USA, 88:11373 (1991)).
The Ras family of proteins are important in the signal transduction pathway modulating cell growth. The protein is produced in the ribosome, released into the cytosol, and post-translationally modified. The first step in the series of post-translational modifications is the alkylation of Cys.sup.168 with farnesyl or geranylgeranyl pyrophosphate in a reaction catalyzed by prenyl transferase enzymes such as farnesyl transferase and geranylgeranyl transferase (Hancock, J. F., et al., Cell, 57:1167-1177 (1989)). Subsequently, the three C-terminal amino acids are cleaved (Gutierrez, L., et al., EMBO J. 8:1093-1098 (1989)), and the terminal Cys is converted to a methyl ester (Clark, S., et al., Proc. Nat'l Acad Sci. (USA), 85:4643-4647 (1988)). Some forms of Ras are also reversibly palmitoylated on cysteine residues immediately N-terminal to Cys.sup.168 (Buss, J. E., et al., Mol. Cell. Biol., 6:116-122 (1986)). It is believed that these modifications increase the hydrophobicity of the C-terminal region of Ras, causing it to localize at the surface of the cell membrane. Localization of Ras to the cell membrane is necessary for signal transduction (Willumsen, B. M., et al., Science, 310:583-586 (1984)).
Oncogenic forms of Ras are observed in a relatively large number of cancers including over 50 percent of colon cancers and over 90 percent of pancreatic cancers (Bos, J. L., Cancer Research, 49:4682-4689 (1989)). These observations suggest that intervention in the function of Ras mediated signal transduction may be useful in the treatment of cancer.
Previously, it has been shown that the C-terminal tetrapeptide of Ras is a "CAAX" motif (wherein C is cysteine, A is an aliphatic amino acid, and X is any amino acid). Tetrapeptides having this structure have been shown to be inhibitors of prenyl transferases (Reiss, et al., Cell, 62:81-88 (1990)). Poor potency of these early farnesyl transferase inhibitors has prompted the search for new inhibitors with more favorable pharmacokinetic behavior (James, G. L., et al., Science, 260:1937-1942 (1993); Kohl, N. E., et al., Proc. Nat'l Acad. Sci. USA, 91:9141-9145 (1994); deSolms, S. J., et al., J. Med. Chem., 38:3967-3971 (1995); Nagasu, T., et al., Cancer Research, 55:5310-5314 (1995); Lemer, E. C., et al., J. Biol. Chem., 270:26802-26806 (1995); Lemer, E. C., et al., J. Biol. Chem., 270:26770 (1995); and James, et al., Proc. Natl. Acad. Sci. USA, 93:4454 (1996)).
Recently, it has been shown that a prenyl transferase inhibitor can block growth of Ras-dependent tumors in nude mice (Kohl, N. E., et al., Proc. Nat'Acad. Sci. USA, 91:914-9145 (1994)). In addition, it has been shown that over 70 percent of a large sampling of tumor cell lines are inhibited by prenyl transferase inhibitors with selectivity over non-transformed epithelial cells (Sepp-Lorenzino, I., et al., Cancer Research, 55:5302-5309 (1995)).
A traditional approach to studying enzymatic activity based on protein-protein interactions is via a standard TCA precipitation assay methodology. Bollag, et al., (1996), Protein Methods (2.sup.nd Ed.) Wiley-Liss, Inc., New York, N.Y. In principle, the modification of a substrate can be detected through radioactive tagging of the substrate and its capture onto glass fiber filters. Total protein is captured by TCA precipitation. In a heterogeneous assay system, limitations include the necessity for washing steps, use of corrosive reagents, such as trichloroacetic acid (TCA) and additional steps such as use of adapter plates and the addition of liquid scintillant. This assay format allows for accurate measurements of modified proteins, but is severely limiting for automated high-throughput screening (HTS).